1. Field of the Invention
The present invention relates generally to methods and apparatus for detecting nucleotide incorporation, and more particularly to a method and apparatus for detecting nucleotide incorporation into a growing polynucleotide chain. The invention is particularly well suited for rapid, automated DNA sequencing.
2. Discussion of the Related Art
Deoxyribonucleic acid (DNA) is composed of four different types of bases: adenine (A), guanine (G), cytosine (C), and thymine (T). A base together with a phosphate and a sugar molecule form a nucleotide. In a DNA molecule, the bases are arranged along a sugar-phosphate backbone to form a chain. These chains are often referred to as "DNA strands." Two DNA strands pair up to form a double-stranded DNA molecule. The strands pair up due to hydrogen bonding between complementary bases. The nucleotide composition and order of a given DNA strand are represented in a DNA sequence. For example, "CCGAT" is a DNA sequence.
DNA sequencing is important for a variety of tasks, including basic scientific research, medical studies, diagnostics, and genome projects. For these tasks, rapid DNA sequencing methods are desirable. However, the speed of previous DNA sequencing methods has been significantly limited by several factors including time and labor for subcloning long DNA fragments into sequencing vectors, sequencing chemistries, and DNA fragment separation techniques, as well as sequence data reading.
Current DNA sequencing techniques are based on the generation of a plurality of DNA fragments corresponding to the sequence of a DNA template. One such DNA sequencing technique is disclosed in Maxam and Gilbert, "A New Method for Sequencing DNA," Proc. Nati. Acad. Sci. USA, Vol. 74(2), February 1977, where a DNA template of interest is chemically degraded to produce a plurality of DNA fragments corresponding to the DNA sequence. Another DNA sequencing technique is disclosed in Sanger et. al., "DNA Sequencing with Chain-terminating Inhibitors," Proc. Natl. Acad. Sci. USA, Vol. 74(12), December 1977, where a plurality of terminated DNA fragments complementary to a DNA template are synthesized. Both of these techniques require labeling the DNA fragments with a reporter, such as a radionucleotide label or a fluorescent label, and electrophoretically separating the labeled DNA fragments on a sieving matrix, such as a polyacrylamide. Sieving matrices are semi-porous materials that separate DNA based on the size of the DNA molecule. Typically, the sieving matrices are poured between two glass plates to form a gel. DNA is applied to one end of the gel, an electrical current is applied, and the negatively-charged DNA molecules travel through the gel toward the cathode, with the smallest DNA molecules traveling the farthest.
This process, called gel electrophoresis, has several drawbacks including (1) laborious gel pouring protocols and (2) variability in the gels due to cleanliness of gel plates, fluctuations in ambient temperature, and inconsistent qualities in gel reagents. This variability can alter the quality of the sequence data and even render the sequence data unusable. Furthermore, separating the DNA fragments requires electrophoresing the fragments at a speed slow enough to (1) permit detection of labeled fragments, (2) avoid decomposition of gel, and (3) allow for adequate separation of fragments. These time constraints limit the speed at which DNA sequencing can be performed using electrophoresis-based sequencing methods.
In sum, limitations of previous DNA sequencing techniques include the need for a reporter label, the time required for performing the techniques, and/or the variability in gels. In light of these limitations, alternative DNA sequencing methodologies are needed.
Atomic force microscopes (AFMs) recently have been used to study biomolecules, as described below. An AFM has a tip that is end-mounted on a flexible cantilever. Interactions between the tip and the sample influence the motion of the cantilever, and one or more parameters of this influence are measured to generate data representative of one or more properties of the sample. AFMs can be operated in different modes including contact mode, TappingMode, (Tapping and TappingMode are trademarks of Digital Instruments, Inc.), and non-contact mode. In contact mode, the cantilever is not oscillated, and cantilever deflection is monitored as the probe tip is dragged over the sample surface. In TappingMode, the cantilever is oscillated mechanically at or near its resonant frequency so that the probe tip repeatedly taps the sample surface, thus reducing the probe tip's oscillation amplitude. The oscillation amplitude indicates proximity to the sample surface and may be used as a signal for feedback. U.S. patents relating to Tapping and TappingMode include U.S. Pat. Nos. 5,266,801, 5,412,980, and 5,519,212, by Elings et al., all of which hereby are incorporated by reference. In the non-contact mode, attractive interactions between the probe tip and the sample (commonly thought to be due to Van der Waals' attractive forces) shift the cantilever resonance frequency when the probe tip is brought within a few nanometers of the sample surface. These shifts can be detected as changes in cantilever oscillation resonant frequency, phase, or amplitude, and used as a feedback signal for AFM control.
Whether operating in contact mode, TappingMode, or non-contact mode, feedback is typically used during AFM scanning to adjust the vertical position of the probe relative to the sample so as to keep the probe tip-sample interaction constant. A measurement of surface topography or another sample characteristic may then be obtained by monitoring a signal such as the voltage used to control the vertical position of the scanner. Alternatively, independent sensors may monitor the position of the tip during scanning to obtain a map of surface topography or another measured sample characteristic. Measurements can also be made without feedback by monitoring variations in the cantilever deflection as the probe moves over the surface. In this case, recording the cantilever motion while scanning results in an image of the surface topography in which the height data is quantitative. Additionally, the positioning of the AFM probe can be enhanced by compensating for drift. U.S. patents relating to drift compensation include U.S. Pat. Nos. 5,081,390 and 5,077,473 by Elings et al., both of which are hereby incorporated by reference.
Proposals have been made to use AFMs to study biomolecules. For instance, Radmacher et al., "Direct Observation of Enzyme Activity with the Atomic Force Microscope," Science, Vol. 265, Sept. 9, 1994, (Radmacher) proposes the use of an AFM to measure height fluctuations of an enzyme (lysozyme). Radmacher believed that the measured height fluctuations probably corresponded to motions of lysozyme during hydrolysis of a substrate oligoglycoside.
Other proposed uses of AFM to study biomolecules are disclosed in Hansma, "Atomic Force Microscopy of Biomolecules," J. Vac. Sci. Technol., Vol. B 14(2), March/April 1996 (Hansma). Hansma lists several DNA applications including (1) calculation of persistence lengths for moving DNA molecules, (2) imaging DNA molecules as a nuclease degrades DNA, and (3) monitoring forces between DNA bases.
Still another proposal is disclosed in Kasas et. al., Escherichia coli RNA Polymerase Activity Observed Using Atomic Force Microscopy," Biochemistry, Vol. 36(3), Jan. 21, 1997 (Kasas). Kasas used an AFM to observe an RNA polymerase transcribing DNA templates in sequential AFM images. Kasas also noted that an RNA polymerase can maintain its biological activity when it is adsorbed onto mica.
While Radmacher, Hansma, and Kasas all disclose use of an AFM to study biomolecules, none of these publications disclose using an AFM to detect incorporation of a nucleotide into a growing polynucleotide chain. That is, these publications do not disclose using an AFM to determine the sequence of a DNA molecule.
Determining the sequence of a DNA molecule is, however, contemplated in U.S. Pat. No. 5,620,854 by Holzrichter. Holzrichter discloses use of an AFM to determine the sequence of a DNA template. The Holzrichter patent contains only a very limited discussion of varying concentrations of nucleotides, and has several shortcomings. First, it lacks a sufficient disclosure as to how to determine which of the four nucleotides is incorporated into the growing nascent DNA strand. In particular, Holzrichter proposes that each nucleotide addition reaction is different based on fact that different nucleotide types (e.g., As, Cs, Gs, and Ts) have different base pairing characteristics, and the method can distinguish nucleotides then based on differences in the number of hydrogen bonds. However, only two of four nucleotides have a different number of hydrogen bonds (A and T have two hydrogen bonds and G and C have three hydrogen bonds), and furthermore nucleotides cannot be easily distinguished based on these differences alone. Second, Holzrichter's process makes no correction for background noise. Third, the signal to noise ratio of Holzrichter's process is not high enough to determine which base was incorporated. Thus, Holzrichter does not enable a determination of which nucleotide is incorporated into the DNA template. Therefore, Holzrichter does not enable using an AFM for DNA sequencing or even for distinguishing nucleotide incorporation from other events, such as background movements of the polymerase not related to nucleotide incorporation.